Process for producing high purity isoflavones

ABSTRACT

A process for the production of a high purity isoflavone composition is disclosed. The process involves subjecting plant material to a primary chromatographic step to obtain an isoflavone enriched fraction and subjecting the isoflavone enriched fraction to a second chromatographic step. Also disclosed is a process that involves passing the plant material through an ultrafiltration membrane which has a moleuclar weight cut-off range that produces a plant material permeate prior to subjecting the plant material permeate to primary and secondary chromatographic steps.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional ApplicationNo. 60/371,129, filed Apr. 10, 2002, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a process for producing highpurity isoflavones. More particularly, the present invention relates toa process for producing high purity isoflavones by a process combiningprimary and secondary chromatographic steps.

[0004] 2. Related Art

[0005] Isoflavones are a class of plant flavonoid compounds which haveestrogenic activity. Research has revealed many possible health benefitsthat may be achieved from the consumption of isoflavones. Highcholesterol is believed to contribute to the development of coronaryheart disease and isoflavones have been demonstrated to lowercholesterol in animal studies. Isoflavones may also play a role inpreventing certain types of cancer. Bone loss in women during and aftermenopause may also be prevented or ameliorated by isoflavones. Becauseof these and other potential health effects, the commercial demand forisoflavones is expected to rise rapidly.

[0006] There are a few processes already reported for isoflavonepurification. U.S. Pat. No. 5,679,806 describes a process for purifyingisoflavones from soy molasses by complicated chromatography andcrystallization steps. U.S. Pat. No. 5,919,921 describes a process forpurifying isoflavones from soy molasses by precipitation and drying.U.S. Pat. No. 5,670,632 describes a process based on chromatographyusing strong cation exchange resins. U.S. Pat. No. 6,320,028 describes aprocess for making an isoflavone product from soybeans based onextraction and precipitation. All of these reported processes are toocumbersome for the production of isoflavones on an industrial scale.Also, these processes are unable to make a high purity product. In fact,the typical purity level associated with these methods is only in the 4%to 50% range. For example, while the process disclosed in U.S. Pat. No.6,033,714 produces an excellent isoflavone product from soy molasses,the product is typically from about 30% to about 50% isoflavones on adry solids basis. Therefore, there is a great need for improvedprocesses that can provide very high purity isoflavone products.

SUMMARY OF THE INVENTION

[0007] The present invention provides a simple and effective method toproduce high purity isoflavones from soy solubles. Specifically, thepresent invention provides a process for producing high purityisoflavone fractions from a plant starting material, said processcomprising the steps of subjecting the plant material to a primarychromatographic step to obtain an isoflavone enriched fraction andsubjecting the isoflavone enriched fraction to a second chromatographicstep. The present invention also provides a process for producing highpurity isoflavone fractions from a plant material comprising the step ofpassing the plant material through an ultrafiltration membrane which hasa moleuclar weight cut-off range that produces a plant material permeateand subjecting the plant material permeate to a primary chromatographicstep to obtain an isoflavone enriched fraction and subjecting theisoflavone enriched fraction to a second chromatographic step.

[0008] The present invention also provides a process for separating highpurity isoflavone fractions from an aqueous plant starting material,said process comprising the steps of heating an aqueous plant startingmaterial to a constant temperature selected on a basis of an aqueoussolubility for at least one desired isoflavone fraction that is to berecovered; passing the heated starting material through anultrafiltration membrane to obtain a plant material permeate, themembrane having a cut-off which passes at least one isoflavone fraction;treating the permeate with an adsorptive material; washing theadsorptive material in water; eluting at least one adsorbed isoflavonefraction from the water-washed adsorptive material with aqueous alcoholto obtain an isoflavone enriched fraction; subjecting the isoflavoneenriched fraction to a secondary chromatography with an adsorptivematerial; eluting with one or more series of at leaston bed volume ofaqueous alcohol, at least one isoflavone fraction from the secondarychrmatography; and evaporating the aqueous alcohol from a stream usedduring the elution in order to promote the crystallization of at leastone isoflavone fraction.

[0009] The various objects and advantages of the present invention willbe clear from the description that follows.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The process of the present invention uses the soluble fraction ofsoy materials that results from the processing of soybeans to make soyprotein concentrate. U.S. Pat. No. 4,172,828 contains a detaileddiscussion of a method for processing soy protein from defatted soybeanflakes, and U.S. Pat. Nos. 5,702,752; 5,792,503; and 6,033,714 disclosefurther details of further processing of the soy solubles, also known assoy molasses. Soy solubles, or molasses, is the alcohol-stripped extractthat results from aqueous ethanol extraction of hexane defatted soybeanflakes.

[0011] While the process disclosed in U.S. Pat. No. 6,033,714 providesan excellent product with an isoflavone content in dried materialproduced from soy molasses of from about 30% to about 50% on a drysolids basis, the inventors of the present invention have surprisinglydiscovered that a process utilizing a primary and then a secondarychromatographic step, will produce a high purity isoflavone enrichedfraction that has a purity in a range of about 70% to about 100%. Theinventors of the present invention have also surprisingly discoveredthat a process utilizing an ultrafiltration step followed by a primaryand then a secondary chromatographic step, will produce a high purityisoflavone enriched fraction that has a pruity in a range of about 70%to about 95%. The inventors of the present invention have alsosurprisingly discovered that a process utilizing an ultrafiltration stepfollowed by a primary and then a secondary chromatographic step, willproduce a high purity isoflavone enriched fraction that has a pruity ina range of about 80% to about 95%.

[0012] The inventors of the present invention have also surprisinglydiscovered that a process utilizing an ultrafiltration step followed bya primary and then a secondary chromatographic step, will, afterevaporation and drying of the product fraction from the secondchromatographic step, produce a greater than 90% pure isoflavoneproduct.

[0013] Soy solubles, or soy molasses, is first pH adjusted to a pH offrom about 4 to about 10, preferably from about 4.5 to about 9.5, mostpreferably from about 5 to about 9. As those of skill in the art willrecognize, the optimum pH of this material will depend in large partupon the requirements of the relative amounts of the types and forms ofspecific isoflavones desired in the final product. The temperature ofthe material must also be adjusted, in large part for the same reasons,and is typically within the range of from about 40° C. to about 100° C.,preferably from about 50° C. to 95° C., most preferably about 60° C. toabout 95° C. The molecular weight cut-off (MWCO) of the ultrafiltrationmembrane can vary, typically anywhere from about 600 to about 1,000,000but a preferred MWCO is about 100,000. Alternatively, the membrane canbe selected on the basis of pore size, with about 0.1 micron a preferredsize. (See U.S. Pat. No. 6,033,714). The ultrafiltration system shouldbe set up to run at optimum transmembrane pressure, and in preferredembodiments a diafiltration feed is provided in order to recover agreater percentage of isoflavones in the permeate.

[0014] This ultrafiltration permeate is then subjected to a primarychromatography step. The chromatography may be either batch orcontinuous, and any number of chromatographic resins may be employed inthe process of the present invention. U.S. Pat. No. 6,033,714 listsuseful resin types and discusses “Amberlite” XAD-4 specifically. Theanionic adsorbent resin TULSION A-2X MP (Thermax Inc., Novi, Mich.) isanother useful resin for the practice of the present invention. The pHand temperature of this feed are adjusted to a pH of from about 5 toabout 10, more preferably from about pH 5.5 to about 9.0, and thetemperature of the column can be regulated as necessary and appropriate,using the proper column design and equipment. As above, the temperatureis typically within the range of from about 60° C. to about 95° C. Asthose of skill in the art will recognize, column size and design willdepend in large part upon the amounts of materials being fed andproduced, and can vary widely.

[0015] In addition, of course, the column design and the choice of resinwill dictate, in part at least, the parameters used in running thechromatography (for example the number of rinses, and the number of bedvolumes of alcohol used to elute the isoflavones from the column).Rinsing of the column is typically performed with deionized water;isoflavones are typically eluted from resins such as those used in thepractice of the present invention using aqueous alcohol. For example,ethanol, methanol, or isopropanol may be used, with a preferredpercentage of alcohol of from about 65% to about 100%, and morepreferred from about 67% to about 95%, and even more preferred fromabout 70% to about 90%. An alcohol gradient process for elution may alsobe employed.

[0016] The elution product from this primary chromatography is thensubjected to a secondary chromatography. Similar resins can be employed;specific resins found to be useful in the practice of this secondarychromatography include, for example, the non-ionic adsorbent resinTULSION ADS 600 (Thermax Inc., Novi, Mich.), DOW OPTIPORE SD-2 (DowChemical, Midland, Michigan), DOW OPTIPORE XUS 40325 (Dow Chemical,Midland, Mich.), and Rohm & Haas XAD 1600 or Rohm & Haas XAD 7HP resins.As is the case with regard to the primary chromatography, thepre-treatment of the resin chosen for the secondary chromatography willbe dictated in large part by the manufacturer's specifications, and bythe relative amounts of the various forms and types of isoflavonesdesired in the final product. Furthermore, as above, the pH andtemperature of the feed will be adjusted for the same reasons. Also, thenumber of bed volumes of wash water and eluent will also be a matter ofchoice and process parameter optimization well within the ordinary skillin the art given the instant disclosure. Again, aqueous alcohols are thepreferred eluents for the practice of the present invention. In certainembodiments, a double elution is practiced; the first using a loweralcohol concentration (from about 30% to about 50%), the second using ahigher alcohol concentration (from about 65% to about 80%).

[0017] After the elution(s) from the secondary chromatography, therecovered fraction(s) may be evaporated and dried. Evaporation can beaccomplished by any of a number of art-recognized methods, including,for example, a multi-effect evaporator. Drying is also accomplished byany number of art-recognized methods, including, for example, spraydrying, freeze drying, and the like. A preferred method for drying isspray drying.

EXAMPLE 1 Primary Chromatography

[0018] Experiments were carried out in a jacketed glass columncontaining 100 mls of an anionic adsorbent resin (in this case TULSIONA-2X MP). The temperature of the column was maintained at 60-65° C. bycirculating hot water through the jacket of the column. The resins wereconditioned by running 3 bed volumes (300 mls) of a solution of 2.5%NaOH through the resin. The resins were then rinsed with 3 bed volumesof deionized water. The flow rate for all steps in these tests was 12mls/min. Ultra-filtration permeate, obtained from ADM Nova Soy plant,was used as feed in these tests. The pH of the feed material was 5.5.This feed material (1.5 liters) was then pumped through the resin. Then3 bed volumes of deionized water was run through the column. After therinse, 5 bed volumes (500 mls) of a solution of 90% ethanol was pumpedthrough the resin. The effluent was sampled and collected as PRODUCT.The results from this operation appear below: Isoflavones Dry SolidsSAMPLE (mg/Kg) (g/Kg) Purity Yield FEED 1020.3 57.5 1.8 PRODUCT 4147.97.3 57.1 81.31

EXAMPLE 2 Primary Chromatography

[0019] This experiment was the similar to that described in Example 1with the following exceptions:

[0020] 1. Feed material was pH adjusted to 8.6.

[0021] 2. Feed volume was 20 bed volumes.

[0022] 3. Ethanol solution was of 70% concentration.

[0023] The results from this operation appear below: Isoflavones DrySolids SAMPLE (mg/Kg) (g/Kg) Purity (%) Yield (%) FEED 544.9 47.1 1.2PRODUCT 857.4 1.4 63.0 78.7

EXAMPLE 3 Primary Chromatography

[0024] This experiment was the similar to that described in Example 2with the following exceptions:

[0025] 1. Feed material was pH adjusted to 9.0.

[0026] 2. Resin used was ROHM & HAAS XAD4.

[0027] The effluent streams from this operation appear below:Isoflavones Dry Solids SAMPLE (mg/Kg) (g/Kg) Purity (%) Yield (%) FEED1773.7 203.7 0.9 PRODUCT 5476.7 10.8 50.9 77.2

EXAMPLE 4 Secondary Chromatography

[0028] Experiments were carried out in a jacketed glass columncontaining 100 mls of non-ionic adsorbent resin (in this case TULSIONADS 600). The temperature of the column is maintained at 60-65° C. bycirculating hot water through the jacket of the column. The resins wereconditioned by running 3 bed volumes (300 mls) of a solution of 2.5%NaOH through the resin. The resins were then rinsed with 3 bed volumesof deionized water. The flow rate for all steps in this test was 12mls/min. Ethanol from the product made by the primary chromatography wasused as the starting feed in these tests. This feed was diluted withwater and the pH was adjusted to 9.3. One liter of this feed materialwas then passed thruough the resin bed. Then 3 bed volumes of deionizedwater was passed through the column. After the rinse, 5 bed volumes (500mls) of a solution of 35% ethanol was passed through the resin. Theeffluent was sampled and collected as PROD. FRAC1. Then 5 bed volumes ofa solution of 70% ethanol was passed through the column. The elutedvolume was collected as PROD. FRAC2. The results are indicated below:Isoflavones Dry Solids SAMPLE (mg/Kg) (g/Kg) Purity (%) Yield (%) FEED785.1 1.6 49.1 PROD.FRAC 1 1804.9 1.9 95.0 97.3 PROD.FRAC 2 6.7 0.0100.0 0.4

EXAMPLE 5 Secondary Chromatography

[0029] This experiment was similar to that described in Example 4 withthe following exceptions:

[0030] 1. 5 bed volumes (500 mls.) of 35% ethanol (isocratic elution)were used.

[0031] The results from this operation appear below: Isoflavones DrySolids SAMPLE (mg/Kg) (g/Kg) Purity (%) Yield (%) FEED 785.1 1.6 49.1PROD. FRAC1 1563.6 1.6 97.7 100.6

EXAMPLE 6 Secondary Chromatography

[0032] This experiment was similar to that described in Example 4 withthe following exceptions:

[0033] 1. Resin used was DOW OPTIPORE SD-2.

[0034] 2. Feed volume through column was 20 bed volumes.

[0035] The effluent streams from this operation appear below:Isoflavones Dry Solids SAMPLE (mg/Kg) (g/Kg) Purity (%) Yield FEED 692.81.9 36.5 PROD. FRAC1 1501.2 2.6 57.7 57.0 PROD. FRAC2 1187.7 1.2 99.045.1

EXAMPLE 7 Secondary Chromatography

[0036] This experiment was similar to that described in Example 4 withthe following exceptions:

[0037] 1. Resin used was DOW OPTIPORE XUS 40325.

[0038] 2. Feed volume through column was 20 bed volumes.

[0039] The results from this operation appear below: Isoflavones DrySolids SAMPLE (mg/Kg) (g/Kg) Purity Yield (%) FEED 903.9 2.1 43.0 PROD.FRAC1 1889.7 3.0 63.0 52.3 PROD. FRAC2 1166.0 1.2 97.2 32.3

EXAMPLE 8 Secondary Chromatography

[0040] This experiment was similar to that described in Example 4 withthe following exceptions:

[0041] 1. Resin used was ROHM & HAAS XAD 1600.

[0042] 2. Feed volume through column was 19 bed volumes.

[0043] The effluent streams from this operation appear below:Isoflavones Dry Solids SAMPLE (mg/Kg) (g/Kg) Purity Yield (%) FEED1067.9 1.6 66.7 PROD. FRAC1 3759.6 4.2 89.5 92.7 PROD. FRAC2 360.4 0.490.1 8.8

EXAMPLE 9 Secondary Chromatography

[0044] This experiment was similar to that described in Example 4 withthe following exceptions:

[0045] 1. Resin used was ROHM & HAAS XAD 7HP.

[0046] 2. Feed volume through column was 20 bed volumes.

[0047] The effluent streams from this operation appear below:Isoflavones Dry Solids SAMPLE (mg/Kg) (g/Kg) Purity Yield FEED 981.8 2.049.1 PROD. FRAC1 2866.4 5.0 57.3 73.0 PROD. FRAC2 701.7 1.0 70.2 17.9

EXAMPLE 10 Ultra-Filtration

[0048] Soy solubles from ADM Soy Protein Concentrate plant were used asthe staring material for isoflavones purification. 31 liters of thisfeed material were pH adjusted to 9.3 and heated to 77° C. This materialwas then ultra-filtered using a 100,000 MWCO polymeric membrane untilthere was 6 liters of retentate left in the feed tank. The filtrationsystem was run in such a way as to maintain a 22-24 p.s.i. transmembranepressure. Deionized water was added to the feed tank to a volume of 31liters. This diafiltration feed was then run through the same systemuntil there was 6 liters of retentate left in the tank. The recordedcomponents of the resulting streams are indicated below: Isoflavones DrySolids SAMPLE (mg/Kg) (g/Kg) Purity (%) Yield (%) FEED 1111.5 83.0 1.34PERMEATE 725.5 65.0 1.12 DIAPERMEATE 352.4 20.0 1.76 78.2 RETENTATE1067.2 108.0 0.99

[0049] Having now fully described the present invention in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be obvious to one of ordinary skill in the artthat the same can be practiced by modifying or changing the inventionwith a wide and equivalent range of conditions, formulations and otherparameters thereof, and that such modifications are intended to beencompassed within the scope of the appended claims.

[0050] All publications, patents and patent applications mentioned inthis specification are indicative of the level of skill of those skilledin the art to which this invention pertains, and are herein incorporatedby reference to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

What is claimed is:
 1. A process for producing a high purity isoflavoneenriched fraction from plant material, said process comprising the stepsof: (a) subjecting plant material to a primary chromatographic step toobtain an isoflavone enriched fraction; and (b) subjecting theisoflavone enriched fraction of step (a) to a secondary chromatographicstep, thereby producing a high purity isoflavone enriched fraction. 2.The process of claim 1, wherein said high purity isoflavone enrichedfraction has a purity in a range of about 70% to about 100%.
 3. Theprocess of claim 2, wherein said high purity isoflavone enrichedfraction has a purity in a range of about 70% to about 95%.
 4. Theprocess of claim 3, wherein said high purity isoflavone enrichedfraction has a purity in a range of about 80% to about 95%.
 5. Theprocess of claim 4, wherein said high purity isoflavone enrichedfraction has a purity of about 90%.
 6. The process of claim 1, whereinsaid plant material is aqueous.
 7. The process of claim 1, wherein saidplant material is selected from a group consisting of soy molasses andsoy whey.
 8. The process of claim 1, wherein said plant material isheated.
 9. The process of claim 8, wherein the temperature of said heatis in a range of about 65° C. to about 95° C.
 10. The process of claim1, wherein prior to step (a), said plant material is passed through anultrafiltration membrane which has a molecular weight cut-off range thatproduces a plant material permeate containing at least one isoflavonefraction.
 11. The process of claim 10, wherein said ultrafiltrationmembrane has a nominal molecular weight cut-off in a range of about 66to about 1,000,000.
 12. The process of claim 11, wherein said molecularweight cut-off is about 100,000.
 13. The process of claim 1 wherein saidprimary and said secondary chromatographic steps use an adsorptivematerial which is selected from the group consisting of an ionic resinand a non-ionic resin.
 14. The process of claim 13, wherein said ionicresin is selected from the group consisting of ionic divinyl-benzenecopolymer, ionic ethylvinylbenzene-divinyl-benzene copolymer, and ionicstyrene-divinyl-benzene copolymer, ionic polystyrene.
 15. The process ofclaim 13, wherein said non-ionic resin is selected from the groupconsisting of non-ionic divinyl-benzene copolymer, non-ionicethylvinylbenzene-divinyl-benzene copolymer, non-ionicstyrene-divinyl-benzene copolymer and non-ionic polystyrene.
 16. Theprocess of claim 1, wherein said primary chromatographic step is acontinuous process, using a plurality of liquid chromatography columnswherein at least one of said columns is loading while another of atleast one of said columns is washing while another of at least one ofsaid columns is eluting.
 17. The process of claim 1, wherein saidsecondary chromatographic step is a continuous process, using aplurality of liquid chromatography columns wherein at least one of saidcolumns is loading while another of at least one of said columns iswashing while another of at least one of said columns is eluting. 18.The process of claim 1, wherein said secondary chromatographic step isfollowed by a process of evaporation.
 19. The process of claim 18,wherein crystals are produced by said process of evaporation.
 20. Theprocess of claim 18, wherein said process of evaporation is followed bya process selected from the group consisting of decanting, hydrocloning,centrifuging and filtering.
 21. The process of claim 18, wherein solidsare produced by said process of evaporation.
 22. The process of claim21, wherein the range of said solids in said high purity isoflavonefraction is of about 1 to about 20%.
 23. The process of claim 18,wherein said process of evaporation is followed by a process of reverseosmosis.
 24. The process of claim 23, wherein said process of reverseosmosis is carried out at a temperature in a range of about 65° C. toabout 95° C.
 25. The process of claim 23, wherein said process ofreverse osmosis is followed by a process of concentration to produce anisoflavone enriched product.
 26. The process of claim 25, wherein saidisoflavone enriched product has an isoflavone concentration in a rangeof about 40% to about 90% on a dry weight basis.
 27. The process ofclaim 25, wherein said isoflavone enriched product has an isoflavoneconcentration in a range of about 90% to about 100% on a dry weightbasis.
 28. The process of claim 1, wherein said high purity isoflavoneenriched fraction is dried.
 29. The process of claim 28, wherein saiddrying is carried out by a process selected from the group consisting ofspray drying, vacuum belt drying and freeze drying.
 30. The process ofclaim 28, wherein said drying is followed by a process selected from thegroup consisting of centrifuging and filtering.
 31. The process of claim1, wherein said high purity isoflavone enriched fraction is cooled. 32.The process of claim 31, wherein the temperature of said cooling is in arange of about 4° C. to about 45° C.
 33. The process of claim 32,wherein said high purity isoflavone is centrifuged.
 34. The process ofclaim 33, wherein said centrifugation is at about 900× g.
 35. A productcomprising the high purity isoflavone enriched fraction of claim 1.